High throughout sequencing of paired VH and VL transcripts from B cells secreting antigen-specific antibodies

Abstract

Methods for determining the VH:VL antibody repertoire from cells, such as antigen-specific B cells, in a high throughput manner. In some aspects, methods are provided for the capture of mRNA transcripts from single B cells that secreted antigen-specific antibodies followed by sequencing of the corresponding cDNAs encoding antibody VH and VL sequences. Libraries of antibodies produced by such methods are also provided.

What is claimed is: 
     
       1. A method for identifying the sequence of two or more transcripts from a plurality of single cells comprising:
 a) incubating isolated single cells and capture agents in individual compartments, wherein the capture agents bind to both mRNA and antibodies; 
 b) lysing the isolated cells; 
 c) contacting the capture agents, along with bound mRNA and antibodies, with affinity agents that bind to antibodies to form complexes; 
 d) purifying the complexes; 
 e) performing reverse transcription and PCR amplification on individual complexes to generate linked amplification products comprising two or more cDNAs from said cell; and 
 e) sequencing amplification products to identify antibody sequences from a single cell. 
 
     
     
       2. The method of  claim 1 , wherein the plurality of single cells comprise a plurality of individual B cells. 
     
     
       3. The method of  claim 2 , wherein the individual B cells are primary B-cells from a human subject. 
     
     
       4. The method of  claim 1 , wherein the capture agents are beads. 
     
     
       5. The method of  claim 1 , wherein the capture agents comprise oligonucleotides which hybridize mRNA. 
     
     
       6. The method of  claim 1 , wherein said sequencing comprises sequencing antibody VH and VL sequences for an antibody that is bound to an antigen of interest. 
     
     
       7. The method of  claim 6 , wherein the capture agents comprise the antigen of interest. 
     
     
       8. The method of  claim 7 , wherein the capture agents are beads that comprise the antigen of interest and a mRNA binding moiety. 
     
     
       9. The method of  claim 1 , wherein the affinity agents are beads. 
     
     
       10. The method of  claim 9 , wherein the affinity agents comprise polystyrene beads. 
     
     
       11. The method of  claim 9 , wherein the affinity agents are magnetic beads. 
     
     
       12. The method of  claim 1 , wherein the affinity agents have density (mass per unit volume) that is less than the capture agents. 
     
     
       13. The method of  claim 1 , wherein the affinity agents bind to an antibody constant region. 
     
     
       14. The method of  claim 1 , wherein the individual compartments are wells in a gel or microtiter plate. 
     
     
       15. The method of  claim 1 , wherein the compartments are sealed with a permeable membrane prior to lysing the individual cells. 
     
     
       16. The method of  claim 1 , wherein lysing the isolated cells comprises the use of a mild detergent solution. 
     
     
       17. The method of  claim 1 , comprising identifying antibody sequences from at least 10,000 individual cells. 
     
     
       18. The method of  claim 1 , wherein purifying the complexes comprises separating complexes by magnetic properties or by density. 
     
     
       19. The method of  claim 1 , wherein step (e) comprises performing emulsion-reverse transcription and PCR amplification on individual complexes.